Polymerase Chain Reaction-Dynamic Light Scattering Sensor for DNA and Protein by Using Both Replication and Cleavage Properties of Taq Polymerase.

The incorporation of AuNPs into polymerase chain reaction (PCR) has become a promising strategy to develop sensitive sensing platforms, due to desirable optical properties of AuNPs and the exponential amplification power of PCR. However, the combination of AuNPs to PCR usually fails to reach expected sensitivity along with additional steps. Here we report a one-step and universal PCR-based biosensor for size-dependent detection of nucleic acids and proteins by using the dynamic light scattering (DLS) technique. This sensing system employs a DNA modified AuNPs (AuNPs-DNA) probe to serve as the TaqMan like signal probe, in which DNA on the surface of AuNPs can be cleaved by Taq polymerase during the extension process of PCR, causing the aggregation of AuNPs to be detected by DLS. This strategy enables sequence-specific recognition of target double-stranded DNA (dsDNA), overcoming the background signal change that triggered by the increase of the PCR cycle. It further demonstrates its applicability in detecting a foodborne pathogen Listeria monocytogenes with a detection limit of 1.2 fg µL-1, which is more sensitive than colorimetric methods. Moreover, by utilizing a proximity assay strategy, this biosensor shows the capability to the homogeneous detection of protein, showing a detection limit of 1.0 pM for a model thrombin. Together, the work demonstrates a reliable strategy to incorporate AuNPs into PCR amplification process as a signal probe, instead of the post-PCR process.

RGB color analysis of formaldehyde in vegetables based on DNA functionalized gold nanoparticles and triplex DNA.

A highly sensitive and selective RGB color analysis for the detection of formaldehyde (FA) was developed by using a DNA functionalized gold nanoparticle (AuNPs-DNA) probe. When complementary oligonucleotides (oligo 2 and oligo 3) and a silver ion (Ag+) were added to the AuNPs-DNA solution, triplex DNA was formed, resulting in the aggregation of AuNPs, and accompanied by a solution color change from red to purple. With the addition of formaldehyde, it reacted with Ag+, decreased the stability of triplex DNA between AuNPs-DNA, induced the dispersion of AuNPs, and the color of AuNPs recovered to red. Therefore, the formaldehyde concentration could be estimated with the RGB (red, green, blue) values of the AuNP solution by using a smartphone application (APP). The R value of the system was proportional to the concentration of formaldehyde within the range of 0.23-4.50 mg L-1, with a detection limit of 0.14 mg L-1. The method has been successfully applied to detect the residues of formaldehyde in vegetable samples and has the potential of the on-site determination of formaldehyde.

Dynamic light scattering and fluorescence dual-signal sensing of cancer antigen-125 via recognition of the polymerase chain reaction product with gold nanoparticle probe.

Cancer antigen 125 (CA - 125) is an important biomarker for the diagnosis of ovarian cancer. In this paper, oligonucleotide 5'-GACAGGCCCGAAGGAATAGATAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3' (oligo 1) contains an aptamer of CA - 125, and was designed partly complementary to oligonucleotide 5'-CTCTCTCTCCACCTTCTTCTTTGAGCGTTTATTCTTGTCT-3' (oligo 2). Oligo 1 · oligo 2 was extended with the Klenow fragment (exo-) polymerase for further polymerase chain reaction (PCR) processes in the presence of two primers: deoxyribose nucleoside triphosphate and Taq polymerase. Single-stranded DNA was produced at two sides of the PCR product by introducing a C18 spacer into the two primers, which could hybridize with AuNPs-DNA probes, investigated by dynamic light scattering and fluorescence. The addition of CA - 125 can interrupt the hybridization between oligo 1 and oligo 2, causing the average diameter of AuNPs-DNA probes to decrease with the increase of CA-125 within the range of 5 fg mL-1 - 50 ng mL-1. The linear regression equation of this relationship was D = 430.48-49.60 log10C, with a detection limit of 1.1 fg mL-1. Fluorescein molecules were modified at the end of the forward primer. The fluorescence intensity of the PCR product can be measured simultaneously, with the fluorescence intensity increasing linearly with the logarithm of CA-125 concentration within a linear range from 10 fg mL-1 to 50 ng mL-1, with a detection limit of 1.5 fg mL-1.

A novel label-free fluorescent detection of histidine based upon Cu2+-specific DNAzyme and hybridization chain reaction.

A novel label-free fluorescent sensor for histidine was developed based upon Cu2+-specific DNAzyme, hybridization chain reaction(HCR) and triplex DNA. Cu2+ can bind to the histidine, in the presence of histidine, leading to the inhibition of the cleavage of substrate strand of Cu2+-dependent DNAzyme, then the intact substrate strand trigger the HCR between H1 and H2. The HCR product can be recognized by triplex-forming oligonucleotide (TFO) through triplex formation and reported by the fluorescence of berberine, the fluorescence intensity of the sensing system was proportional to the concentration of histidine during the range of 5.7-455 nmol L-1, with a detection limit of 2.0 nmol L-1.

The peroxidase-mimicking function of acetate and its application in single-enzyme-based glucose test paper.

Acetate ion was widely used in pH buffer to control pH environment. Here we firstly found that acetate ion had mimic peroxidase activity. Acetate ions are capable of catalyzing the decomposition of hydrogen peroxide and play a similar role to that of horseradish peroxidase (HRP). Acetate catalyzes the oxidation of tetramethylbenzidine (TMB) by H2O2, which is the product of the reaction of glucose and glucose oxidase. A colorimetric sensor for H2O2 and glucose was developed using acetate ions. The linear regression equation for H2O2 was A = 0.0029 C + 0.0530 (C (µmolL-1), R = 0.9978), and the detection limit was 3.0 µmolL-1, whereas that for glucose was A = 0.0021 C + 0.0709 (C (µmol L-1), R = 0.9977), and the detection limit was 4.0 µmol L-1. Moreover, the proposed method was successfully applied for the detection of H2O2 in human urine and glucose in human serum; thus, the proposed method could be used for the diagnosis of illness or disease. A single-enzyme-based glucose test paper was firstly prepared and tested for semi-quantitative analysis of glucose.

Strand displacement amplification-coupled dynamic light scattering method to detect urinary telomerase for non-invasive detection of bladder cancer.

Despite huge successes achieved by strand displacement amplification (SDA) and gold nanoparticles (AuNPs) in biomolecules sensing, the strategy of combination of SDA and AuNPs-based dynamic light scattering (DLS) for a biomolecule sensing is unexplored. Here we developed a non-invasive, SDA-based DLS method for the diagnosis of bladder cancer by detecting telomerase activity in human urine. In the presence of telomerase, the telomerase substrate (TS) primer was elongated with repeating sequences of (TTAGGG)n, and the resulting product triggers SDA between the hairpin deoxyribonucleic acid (DNA) and the Primer. The SDA product can be recognized by the oligonucleotide-modified AuNPs probes, resulting in DLS measurable AuNPs aggregation. The assay displayed a detection limit of 3 MCF-7 cells with a signal-to-noise ratio of 3 in a dynamic range of 5-1000 cells. The method was simple, reliable and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. Moreover, only urine samples from bladder cancer patients induced a significant change in the average hydrodynamic diameter, indicating practical applicability of the method for the non-invasive diagnosis of bladder cancer.

Sensitive DNA detection by polymerase chain reaction with gold nanoparticles.

We developed a novel strategy for rapid colorimetric detection of specific DNA sequence based on gold nanoparticles assemblies induced by polymerase chain reaction (PCR) product. In the presence of target DNA, the two DNA-functionalized AuNP probes selectively hybridized with the prohibited nucleic acid segments of two primers owing to the zipping off of the hairpin structures during PCR process, resulted in the aggregation of AuNPs with a concomitant color change from red to blue-purple. It is a convenient and universal method for sensitive DNA detection with no need for any further post-treatment of the PCR products. Most importantly, our method showed a low limit of detection (LOD) of 4.3 fM with a wide range of target DNA from 16 fM to 1.6 nM. Owing to the versatility and low cost, the proposed strategy could be extremely useful for a wide range of applications, providing a promising tool for rapid disease diagnostics and gene sequencing.

A label-free light-up fluorescent sensing platform based upon hybridization chain reaction amplification and DNA triplex assembly.

A label-free light-up fluorescent sensing strategy using hybridization chain reaction (HCR) amplification and DNA triplex assembly has been developed. Remarkably, the proposed fluorescence assay is successfully applied to the determination of avian influenza A (H7N9) virus DNA and thrombin. Herein, in the presence of targets, the target DNA/initiator triggers a cascade of hybridization events between H1 and H2 that yields nicked double helices analogous to alternating copolymers. With the additions of triplex-forming oligonucleotide (TFO) and berberine, the triplex structures form between HCR products and TFO. Then, a large amount of berberine can bind to the triplex structures and the sensing system exhibits a dramatic increase in the fluorescence intensity at 530 nm. Under optimal conditions, the label-free fluorescent sensing platform shows sensitive responses to H7N9 virus DNA and thrombin in the range of 0.2-100 nM and 0.5-200 nM, respectively. The detection limits of H7N9 virus DNA and thrombin are as low as 0.14 nM and 0.32 nM, respectively. Owing to the simplicity and low cost, the proposed strategy can be used in other biomarkers assays, providing a promising tool for clinical diagnosis and biomedical detection.